Growth, development and differentiation of higher organisms is controlled by a highly ordered set of events called the cell cycle (Morgan, 1997). Cell division and cell growth are operated by the cell cycle which ensures correct timing and high fidelity of the different transition events involved. Transition control through and between the different stages of the mitotic cell cycle depend on the activity of cyclin-dependent kinases (CDKs) and their specific subset of cyclins and appears to be conserved in all higher eukaryotes (as the enzymes responsible for DNA replication, the cytoskeleton components that mediate spatial organization within and directed movements of the cell or its contents and the ubiquitin-dependent pathway for the degradation of proteins).
In multicellular eukaryotes, the association of multiple CDKs with different classes of cyclins called mitotic and G1 cyclins allows for the formation of various protein kinase complexes, each required for specific regulatory steps during the cell cycle.
The understanding of the cell cycle progression remains far better in the yeast and mammal model systems from which it was further elucidated that CDK activity is additionally regulated by factors including CDK kinases like the yeast Wee1-type kinases, CDK phosphatases like yeast CDC25, CDK inhibitors like the yeast SIC1 and the human INK4 gene products, and CDK activating kinase (CAK). Cell cycle regulation at both G1→S and G2→M phase transitions depends on the appropriate CDK-cyclin complexes; both transitions are believed to be the major control points in the cell cycle. The cell's decision to proliferate and synthesize DNA and ultimately to divide is made at the G1→S restriction point in late G1. Overcoming this point of no return needs the cell's competence to initiate DNA synthesis as well as the expression of S-phase genes. Transcription of S-phase specific genes requires binding to the DNA of an E2F transcription factor. The heterodimeric E2F/dimerization partner (DP) transcription factor regulates the promoter activity of multiple genes, which are essential for DNA replication and cell cycle control (Helin, 1998; Müller and Helin, 2000). E2F/DP activity is inhibited by the retinoblastoma gene product (Rb) that is regulated by phosphorylation (Weinberg, 1995). E2F transcription factors are critical effectors of the decision to pass the restriction point and to allow the cell to proceed in S-phase.
In plants, post-embryonic development relies on iterative cell division in the meristems. Cells in the meristem remain in an indeterminate state whereas upon differentiation they exit the cell cycle and move from the meristem. In the plant's meristematic or undifferentiated cell system the G1→S transition is characterized by the action of CDK-cyclin complexes involving D-type cyclins. Similar with the mammalian system, phosphorylation of the retinoblastoma protein by the CycD-CDK complex is required to release the associated E2F transcription factor, thereby enforcing the cell's commitment to S-phase and thus the cell's decision to pass over the cell cycle exit point. Thus, plant E2F and DP genes have been identified suggesting their involvement in the G1→S regulatory mechanism (Ramirez-Parra et al., 1999; Sekine et al., 1999; Ramirez-Parra and Gutierez, 2000; Magyar et al., 2000). Their role in the plant cell cycle molecular machinery that controls cell cycle exit and differentiation is still largely unknown. Therefore, one of the objects of the present invention is to identify the regulatory capacity on cell cycle progression of E2F transcription factors by modulating their expression in a plant. Modulating expression of these transcription factors allows manipulating the biological processes that they control. It is a further object of the present invention to modulate these biological processes towards particular useful applications in agriculture and horticulture. The invention provides a solution to at least several of the objects above by providing the embodiments described further.